There has been much technical development in recent years on methods for manipulating and copying DNA molecules for use in modern techniques of molecular biology and genetic engineering. A reaction known as the polymerase chain reaction, or PCR, described in U.S. Pat. Nos. 4,683,195 and 4,683,202, has become a standard laboratory method for reproducing, or amplifying, copies of a DNA molecule either from an in vitro product or from a native DNA sequence isolated from a living organism. A variety of methods have been developed for cloning the products of PCR amplification into a plasmid vector for the purposes of making large quantities of DNA analysis, or to express DNA as RNA or protein products by other in vitro and in vivo methods.
One common method for the cloning of the products of PCR reactions involves the use of circular plasmids which can be cut to produce DNA fragments with ends having single 3' dTMP overhangs. A single dAMP overhang present on the DNA insert can be conveniently ligated into a vector having complementary single dTMP overhangs. These so-called "T-vectors" are formed from two closely spaced sequences that are recognized by a specific restriction enzyme that cleaves the plasmid DNA to leave a single 3' dTMP overhang on each end of the linearized vector following cleavage. The use and construction of such a vector is described in U.S. Pat. No. 5,487,993. The T-vector is intended to take advantage of a phenomenon associated with some of the DNA polymerases that are used in the PCR process. For example, a popularly used DNA polymerase, Taq DNA polymerase, often leaves a single unpaired dAMP residue at each end of the DNA molecules which are the product of the PCR reaction. However, the propensity of Taq DNA polymerase to add single 3' dAMP extensions varies with the composition of the 3' ends of the PCR products, the PCR conditions, and the conditions under which the completed reactions are stored (Hu, DNA Cell Biol. 12, 763 (1993); Magnuson et al., BioTechniques 21, 700, (1996)). For example, the presence of a dAMP residue at the 3' end of a duplex is inhibitory to dAMP addition (presumably leaving a blunt end), and other bases such as dCMP, dGMP or dTMP can be preferentially added to ends having specific sequences. Therefore, under many conditions the fraction of PCR products that possess single 3' dAMP extensions on both ends can be relatively low. Therefore, vectors which have only a single 3' dTMP overhang cannot efficiently ligate with these other fragments. Also, while the theoretical cloning efficiency of a single T overhang and a single A overhang is high, practical cloning efficiencies are sometimes lower, and there can be high numbers of false positives arising from vector religation, due to damage of the DNA ends caused by exonuclease contamination of the restriction enzyme, or religation of the small vector fragment produced by restriction enzyme cleavage instead of the PCR product.
There are other methods for preparing T vectors including the addition of single dideoxy TMP residues at each end of a blunt and linearized plasmid using terminal transferase Holton & Graham, Nucleic Acids Res. 19:1156 (1991), or the addition of linkers containing a single unpaired dTMP residue to a linearized plasmid. However, no method has been described for the preparation of vectors having bases other than dTMP as the single 3' overhang.
Technology generally desires cloning vectors which optimize or improve efficiency over other vectors then currently available.